Degradable branched polymers for biomolecule delivery

Abstract

Disclosed are branched poly(beta-amino ester) (PBAE) comprising a backbone having a ratio of diacrylate monomers and triacrylate monomers or diacrylate and tetraacrylate monomers, an amine sidechain, and an amine endgroup, and a nucleic acid, which are useful for the delivery of biomolecules, including the intracellular delivery of nucleic acids, which can be applied to almost any disease, ranging from cancer, to autoimmune diseases, to ocular diseases, to genetic disorders. These materials are particularly advantageous and enabled for delivery of mRNA and DNA to cells, including human cells. Compared to other PBAE nanoparticles, the disclosed NPs are smaller, more monodisperse, and more efficient at delivery and are useful and translatable as therapeutics.

Description

DEGRADABLE BRANCHED POLYMERS FOR BIOMOLECULE DELIVERY CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 678,277, filed August 1, 2024, which is incorporated herein by reference in its entirety. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant numbers EB028239, CA228133, and CA246699, awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0003] Gene therapy continues to gain increased prominence in medicine with therapeutics reaching the clinic targeting a wide array of disease areas, Arabi et al., 2022, yet effective and safe nucleic acid delivery remains a challenge for many gene-based therapeutics. Nucleic acid-loaded polymeric nanoparticles (NPs) carry several advantages over traditional viral vectors, including low immunogenicity, ease in manufacturability, and large cargo capacities. Nayerossadat et al., 2012. Polymeric gene delivery, however, has not progressed as far in clinical trials as viral gene therapy and lipid nanoparticles have, with ongoing work aiming to improve the efficiency and safety of these technologies. SUMMARY

[0004] In some aspects, the presently disclosed subject matter provides a nanoparticle comprising a branched poly(beta-amino ester) (PBAE) comprising a backbone having a ratio of diacrylate monomers and triacrylate monomers or diacrylate and tetraacrylate monomers, an amine sidechain, and an amine endgroup, and a nucleic acid.

[0005] In certain aspects, the diacrylate monomer is selected from:

[0006] ;9); ); g of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0015] In particular aspects, the diacrylate monomer is:or te monomer is selected from: ;of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0022] In particular aspects, the triacrylate monomer or tetraacrylate monomer is selected from:OH(S3);OH(S4);

[0026] .

[0027] sidechain monomer is selected from: O O (S91).NH2 (E1); NH2 (E2);;OH

[0030] ;[; and

[0032] .

[0033] (E39); and H N N

[0034] NH2(E63).

[0035] In more particular aspects, the branched PBAE is selected from 9,8-91-39; 9,12-91-39; and 9,8-91-39. and siRNA. acid weight-to-weighta a range from about 1% to about 60%.

[0039] In certain aspects, the nanoparticle has an acrylate:amine (Ac:Am) ratio of 2.2:1 or 2.4:1.

[0040] In certain aspects, the nanoparticle has a size between about 10 nm to about 150 nm in diameter. In particular aspects, the nanoparticle has a size of about 50 nm.

[0041] In other aspects, the presently disclosed subject matter provides a method for treating a disease, condition, or disorder, the method comprising administering a therapeutically effective amount of a nanoparticle disclosed herein to a subject in need of treatment thereof. In particular aspects, the disease, condition, or disorder is selected from a cancer, an autoimmune disease, an ocular disease, and a genetic disease or disorder. In more particular aspects, the cancer comprises melanoma or hepatocellular carcinoma.

[0042] In other aspects, the presently disclosed subject matter provides a method for transfecting one or more cells, the method comprising contacting the one or more cells with a nanoparticle disclosed herein.

[0043] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below. BRIEF DESCRIPTION OF THE FIGURES

[0044] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0045] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0046] FIG. 1A is a schematic of an overview of the presently disclosed subject matter.

[0047] FIG. 1B shows the representative structures and synthesis scheme. Linear PBAEs contain a backbone synthesized via polymerization of a diacrylate monomer and amine side chain. Branched PBAE (BPBAE) backbones contain a ratio of diacrylate and triacrylate monomers (trifunctional BPBAE) or diacrylate and tetraacrylate monomers (tetrafunctional BPBAE) polymerized with an amine side chain.

[0048] FIG.2A, FIG.2B, FIG.2C, and FIG.2D demonstrate B8-based BPBAE NP evaluation for efficacy and cytotoxicity. B16F10 murine melanoma cells and ARPE-19 human retinal pigment epithelial cells were transfected with GFP DNA-loaded BPBAE NPs. (FIG. 2A) Screened backbone, side chain, and end-capping monomers used (n = 1.5 in B9). (FIG. 2B) Various BPBAEs led to robust GFP transfection depending on polymer structure, with 9,8-91-39 leadingto the highest transfection in either cell line. (FIG. 2C) Transfection efficacy correlated strongly between the two cell types. (FIG. 2D) All BPBAEs maintained high cellular viability (relative metabolic activity). B9, S91, and E39 are used in all subsequent experimentation. Significance assessed via one-way ANOVA with Dunnett’s posttest comparing all conditions to 7,8-4-6. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant.

[0049] FIG. 3A and FIG. 3B demonstrate assessment of branching monomer, branching percentage, and acrylate:amine (Ac:Am) feed ratio on BPBAE transfection efficacy. BPBAEs were synthesized using B9, S91, E39 and either B8, B12, or B13 at 0,10, 20, 30, 40, 50, and 60% branching, with Ac:Am ratios of 2.2:1 and 2.4:1. (FIG. 3A) Triacrylate (B8) and tetraacrylate (B12, B13) monomers used to generate branched structures. (FIG. 3B) Transfection of B16F10 murine melanoma cells with GFP DNA-loaded NPs. GFP transfection measured as %GFP+ cells (left) and mean fluorescence intensity (right) using 25 (top) and 50 (bottom) w / w NPs. Error bars are SEM.

[0050] FIG. 4A, FIG. 4B, and FIG. 4C demonstrate the effect of percent branching and polymer:DNA weight-to-weight (w / w) ratio on BPBAE NP transfection efficacy and cytotoxicity. B16F10 murine melanoma cells were transfected with GFP DNA-loaded NPs at 10, 20, and 40 w / w. (FIG. 4A) %GFP+ and GFP MFI for cells transfected with 9,8-91-39 (left), 9,12-91-39 (middle), and 9,13-91-39 (right). (FIG. 4B) Representative 20× images showing GFP expression (green). Scale bars are 100 μm. (FIG. 4C) Cell viability (relative metabolic activity) with significance assessed via one-way ANOVA with Dunnett’s posttest comparing all conditions to UT. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant.

[0051] FIG.5A, FIG.5B, FIG.5C, FIG. 5D, FIG. 5E, FIG.5F, FIG.5G, FIG.5H, FIG.5I, and 5J show the biophysical characterization of 20 w / w linear (0%), moderately branched (30%), and highly branched (60%) PBAE NPs. (FIG. 5A) All formulations utilized PBAE 9,X-91-39 at various degrees of branching. (FIG. 5B) The DNA gel electrophoresis binding assay showed complete complexation with all NP formulations, with no free DNA available to run in the gel. (FIG.5C) Surface charge (zeta potential) of PBAE NPs was assessed via electrophoretic mobility. (FIG. 5D) Intensity-weighted Z-average NP hydrodynamic diameter and (FIG. 5E) polydispersity index (PDI) were assessed via dynamic light scattering (DLS). (FIG. 5F) Number-averaged particle size distribution (n = 3 combined replicates per group) and (FIG. 5G) mean NP hydrodynamic diameter were assessed via nanoparticle tracking analysis (NTA). (FIG. 5H)Estimated average number of plasmid molecules per NP and corresponding (FIG. 5I) distribution (n = 3) were calculated by NTA. (FIG. 5J) DNA release assay using heparin to compete off DNA from the NPs demonstrated tighter binding affinity with branched polymers.

[0052] FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D show the transfection of labeled DNA reveals kinetics of NP uptake based on PBAE branching. B16F10 cells were transfected with 20 w / w linear and BPBAE NPs loaded with Cy5-labeled GFP DNA. Flow cytometry was performed on cells 30, 60, and 120 min following NP administration. Linear and BPBAE formulations led to high rates of cell internalization (FIG. 6A) and robust overall uptake (FIG. 6B). Significance in (FIG. 6A) and (FIG.6B) assessed via two-way ANOVA with Dunnett’s posttest comparing all conditions to the linear NP formulation (comparisons performed as simple effects at each time point). (FIG.6C) Histograms of Cy5 MFI over time, representing n = 4 concatenated replicates. (FIG. 6D) NP uptake (Cy5 MFI) 120 min following NP dosing correlated strongly with transfection efficiency (GFP MFI) 48 h following transfection. Vertical error bars in (FIG.6A), (FIG.6B), and (FIG.6D), and horizontal error bars in (FIG. 6D) are SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0053] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, and FIG. 7E demonstrate that branched PBAE NPs efficiently deliver pDNA to various cell types, including human and murine cancerous and noncancerous cells. GFP DNA-loaded BPBAE NPs at 20 w / w were administered to (FIG. 7A) ARPE-19 human retinal pigment epithelial cells, (FIG. 7B) A375 human melanoma cells, (FIG. 7C) Hepa1-6 murine hepatocellular carcinoma cells, and (FIG. 7D) NIH / 3T3 murine fibroblast cells. (FIG.7E) Representative 20× images of GFP transfection (green). Cell viability assessed via relative metabolic activity. Scale bars are 100 μm. In A-D, significance assessed via one-way ANOVA with Dunnett’s posttest comparing all conditions to linear control. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant.

[0054] FIG. 8A and FIG. 8B demonstrate that BPBAE NPs successfully deliver mRNA and siRNA. (FIG. 8A) GFP mRNA-loaded NP transfection of B16F10 murine melanoma cells leads to high GFP expression with minimal cellular toxicity. (FIG. 8B) Transfection of B16F10 cells stably expressing GFP with NPs loaded with siRNA against GFP leads to robust GFP knockdown and minimal cellular toxicity. Knockdown assessed relative to scrambled (scrRNA) controls. Histograms in (FIG.8B) represent n = 4 concatenated replicates. Cell viability assessed via relative metabolic activity. Significance assessed via one-way ANOVA with Dunnett’s posttest comparingall groups to linear control. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant.

[0055] FIG.9A, FIG.9B, FIG.9C, and FIG.9D show the flow cytometry gating strategy for (FIG. 9A) GFP transfections and (FIG.9C) Cy5-labeled DNA uptake assay. GFP+ and Cy5+ populations were gated relative to untreated controls. (FIG. 9B, FIG. 9D) Example treated samples. GFP MFI and Cy5 MFI are reported as BL1-A and RL1-A intensities as fold over untreated, respectively. The same strategy used in (FIG. 9A) is performed for GFP mRNA transfections. For siRNA transfections, %Knockdown is reported as GFP MFI relative to scrRNA controls.

[0056] FIG 10 is GPC traces of 2.4:1 Ac:Am BPBAEs. Legend refers to branching monomer and branching degree (0%=linear 9-91-39). Note: y-axis is reversed to improve readability.

[0057] FIG.11A and FIG.11B.1H NMR of B9 diacrylate and B8 triacrylate backbone monomers. (FIG. 11A) B9, n=1.5. (FIG. 11B) B8.

[0058] FIG. 12A and FIG. 12B are1H NMR of B12 and B13 tetraacrylate backbone monomers. (FIG. 12A) B12. (FIG. 12B) B13.

[0059] FIG. 13A and FIG. 13B are1H NMR of S91 sidechain and E39 endcap monomers. (FIG. 13A) S91. (FIG. 13B) E39.

[0060] FIG. 14A and FIG. 14B are1H NMR of linear PBAEs. (FIG. 14A) 9-91-392.2:1. (FIG. 14B) 9-91-39 2.4:1. Relevant peaks needed to evaluate branching: diacrylate B9 phenyl (blue). Note that no peaks are observed between 5.83 and 6.41 ppm, indicating a lack of acrylate groups and successful polymer endcapping. Additional peaks from residual diethyl ether.

[0061] FIG.15A and FIG.15B are B8 30% triacrylate PBAEs. (FIG.15A) 9,8-91-3930% 2.2:1. (FIG. 15B) 9,8-91-39 30% 2.4:1. Relevant peaks needed to evaluate branching: diacrylate B9 phenyl (blue) and triacrylate B8 methyl (red). Note that no peaks are observed between 5.83 and 6.41 ppm, indicating a lack of acrylate groups and successful polymer endcapping. Additional peaks from residual diethyl ether.

[0062] FIG.16A and FIG.16B are triacrylate PBAEs. (FIG.16A) 9,8-91-39 60% 2.2:1. (FIG. 16B) 9,8-91-39 60% 2.4:1. Relevant peaks needed to evaluate branching: diacrylate B9 phenyl (blue) and triacrylate B8 methyl (red). Note that no peaks are observed between 5.83 and 6.41 ppm, indicating a lack of acrylate groups and successful polymer endcapping. Additional peaks from residual diethyl ether.

[0063] FIG. 17A and FIG. 17B are1H NMR of B1230% tetraacrylate PBAEs. (FIG. 17A) 9,12- 91-3930% 2.2:1. (FIG.17B) 9,12-91-3930% 2.4:1. Note that no peaks are observed between 5.83 and 6.41 ppm, indicating a lack of polymer endcapping. Additionalpeaks from residual diethyl ether.

[0064] FIG. 18A and FIG. 18B are1H NMR of B1260% tetraacrylate PBAEs. (FIG. 18A) 9,12- 91-3960% 2.2:1. (FIG.18B) 9,12-91-3960% 2.4:1. Note that no peaks are observed between 5.83 and 6.41 ppm, indicating a lack of polymer endcapping. Additionalpeaks from residual diethyl ether.

[0065] FIG. 19A and FIG. 19B are1H NMR of B1330% tetraacrylate PBAEs. (FIG. 19A) 9,13- 91-3930% 2.2:1. (FIG.19B) 9,13-91-3930% 2.4:1. Relevant peaks needed to evaluate branching: diacrylate B9 phenyl (blue) and tetraacrylate B13 methyl (red). Note that no peaks are observed between 5.83 and 6.41 ppm,groups and successful polymer endcapping. Additional peaks from residual diethyl ether.

[0066] FIG. 20A and FIG. 20B are1H NMR of B1360% tetraacrylate PBAEs. (FIG. 20A) 9,13- 91-3960% 2.2:1. (FIG.20B) 9,13-91-3960% 2.4:1. Relevant peaks needed to evaluate branching: diacrylate B9 phenyl (blue) and tetraacrylate B13 methyl (red). Note that no peaks are observed between 5.83 and 6.41 ppm,groups and successful polymer endcapping. Additional peaks from residual diethyl ether.

[0067] FIG.21 shows the BPBAE NP toxicity dose-response. Cellular viability (relative metabolic activity) of B16F10 murine melanoma cells when dosed with 20 w / w 9,X-91-39 GFP DNA-loaded BPBAEs. Legend refers to branching monomer and branching degree (0%=linear 9-91-39). Dashed line=50% viability.

[0068] FIG.22A, FIG.22B, and FIG.22C demonstrate transfection with high MW linear PBAEs. B16F10 murine melanoma cells were transfected with 20 w / w GFP DNA-loaded 9-91-39 NPs. PBAEs were synthesized at an Ac:Am ratio of 2.1:1 and monomer concentration of 200 or 400 mg / mL prior to endcapping. See Table 3 for MW characterization. (FIG. 22A) %GFP+. (FIG. 22B) GFP MFI. (FIG.22C) Cellular viability assessed via relative metabolic activity. Significance assessed via one-way ANOVA with Dunnett’s posttest comparing all conditions to untreated (UT) control. Error bars are SEM. *P<0.05, **P<0.01, ***P<0.001, ns=not significant.

[0069] FIG. 23A, FIG. 23B, and FIG. 23C show the characterization of 10 and 40 w / w DNA- loaded 9,X-91-39 BPBAE NPs. (FIG. 23A) NP surface charge (zeta potential) assessed viaelectrophoretic mobility. (FIG. 23B) Intensity-weighted Z-average NP diameter and (FIG. 23C) polydispersity index (PDI) assessed via dynamic light scattering (DLS).

[0070] FIG.24A, FIG.24B, and FIG.24C show: (FIG.24A) Surface charge (zeta potential), (FIG. 24B) NP size, and (FIG. 24C) polydispersity index (PDI) of highest-performing RNA-loaded BPBAE NP (9,13-91-3930%) assessed via electrophoretic mobility and dynamic light scattering (DLS).

[0071] FIG. 25 shows the visualization of representative PBAE formulations. PBAEs were formulated as described herein at 20 w / w and imaged via transmission electron microscopy (TEM) with negative staining. Two representative images are shown per formulation. Scale bar = 500 nm.

[0072] FIG. 26 shows highly efficient BPBAE-mediated mRNA delivery to human retinal pigment epithelial cells in vitro. ARPE-19 cells (10k / well) were transfected with 9,8-91-39 (30%, 2.4:1) or canonical linear PBAE 4-5-7 NPs loaded with GFP mRNA. Doses and w / w are noted in the axis labels. UT = untreated control. Error bars are SEM.

[0073] FIG. 27A, FIG. 27B, FIG. 27C, and FIG. 27D show in vitro PBAE screening in Madin- Darby Canine Kidney (MDCK) cells. (FIG. 27A) Monomers tested. (FIG. 27B) Transfection efficacy assessed via fLuc DNA-loaded NPs. (FIG.27C) Cell viability (relative metabolic activity) assessed via GFP DNA-loaded NPs. (FIG. 27D) 10× images (Green = GFP; scale bar = 250 µm) of highest performing condition of top two polymer formulations. Significance assessed via one- way ANOVA with Dunnett’s posttest comparing all conditions to untreated (UT) control. Cells seeded at 5k / well. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant.

[0074] FIG. 28A, FIG. 28B, and FIG. 28C demonstrate development of nanoplasmid-loaded lyophilized BPBAE NPs. NPs were complexed with nanoplasmid GFP DNA and polymer 9,8-91- 39 at 20 w / w and lyophilized, using sucrose as an excipient. NPs were complexed to have a final DNA concentration after reconstitution in water of 0.033 µg / µL (low dose, LD) or 0.1 µg / µL (high dose, HD) and final sucrose concentration of 100 µg / µL. (FIG.28A) NP size, polydispersity index (PDI), surface charge, and osmolarity following reconstitution. (FIG. 28B) Reconstituted NPs successfully transfect MDCK cells in vitro (10× images, Green = GFP; scale bar = 250 µm, 5k cells / well, %Viability assessed via CTG). (Fig. 28C) Reconstituted NPs successfully transfect ARPE-19 cells in vitro (10k cells / well, transfection assessed via flow cytometry, %Viability assessed via CTG). UT = untreated control. Error bars are SEM.

[0075] FIG. 29A and FIG. 29B demonstrate the development of BPBAEs synthesized in a single step. (FIG. 29A) B9 and S91 were reacted using the BPBAE backbone reaction previously described here, with Ac:Am ratios of 1.5:1 to 1.9:1 (legend) followed by ether purification and DMSO resuspension as previously described here. This resulted in PBAE 9-91 in a single reaction step. PBAE 9-91 was then complexed with eGFP-N1 plasmid at various w / w (x-axis) and dosed to B16F10 cells in vitro. (FIG. 29B) B9, B8, and S91 were reacted using the BPBAE backbone reaction previously described herein, with an Ac:Am ratio of 1.8:1 and various branching percentages (legend) followed by ether purification and DMSO resuspension as previously described here. This resulted in BPBAE 9,8-91 in a single reaction step. BPBAE 9,8-91 was then complexed with eGFP-N1 plasmid at various w / w (x-axis) and dosed to B16F10 cells in vitro.250 ng DNA / well. 5k cells / well. %Viability assessed via CTG. Error bars are SEM. UT = untreated control. DETAILED DESCRIPTION

[0076] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0077] Poly(beta-amino ester) (PBAE) nanoparticles (NPs) hold great promise for nonviral gene delivery in a variety of contexts. Recent studies suggest branched PBAEs (BPBAEs) offer unique advantages over linear counterparts, but the effect of polymer structure has not been well investigated across many chemical constituents. In some embodiments, the presently disclosed subject matter provides library of novel BPBAEs synthesized with tri- and tetrafunctional branching. The effects of BPBAE branching and w / w on nanoparticle transfection efficacy to minimize the required polymer dose for efficacious transfection, nanoparticle physiochemical properties, and the broad applicability of these BPBAEs for nucleic acid delivery to diverse cell types were evaluated, characterized, and assessed.

[0078] Representative branched PBAEs previously disclosed include those disclosed in International PCT Patent Application Publication No. WO2024098053 for POLYMER-NUCLEIC ACID NANOPARTICLES FOR GENE EDITING, to Green, published November 6, 2023; International PCT Patent Application No. WO2021081495 for POLYMERIC NANOPARTICLES FOR INTRACELLULAR PROTEIN DELIVERY, to Green, published April 29, 2021, and WO2020077159 for POLY(Beta-AMINO ESTER) NANOPARTICLES FOR THE NON-VIRAL DELIVERY OF PLASMID DNA FOR GENE EDITING AND RETINAL GENE THERAPY, to Green, published April 16, 2020, each of which is incorporated herein by reference in its entirety.

[0079] For example, WO2020077159 disclosed branched PBAEs of the following generic formula: H O N R'" H NO OR'" R' OO O; nucleic acid sequence encoding a

[0084] each R is independently a diacrylate monomer of the following structure:

[0085] or ;

[0086] wherein Rocomprises a linear or branched C1-C30alkylene chain, which may further comprise one or more heteroatoms or one or more carbocyclic, heterocyclic, or aromatic groups and X1 and X2 are each independently a linear or branched C1-C30 alkylene chain;

[0087] each R* is a triacrylate, quanternary, or hexafunctional acrylate monomer selected from the group consisting of: ;

[0091] each R" is independently a side chain monomer comprising a primary, secondary, or tertiary amine; and

[0092] each R'" is independently an end group monomer comprising a primary, secondary, or tertiary amine.

[0093] Likewise, WO2024098053 disclosed branched PBAEs of the following general formula: ;

[0096] m and n are each independently an integer from 1 to 10,000;

[0097] m1 is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0098] m2 is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20;

[0099] q is an integer selected from 0 or 1;

[0100] wherein:

[0101] –(CH2)m1-(C=C)q-(CH2)m2-CH3comprises a hydrophobic sidechain;

[0102] R comprises a divalent radical comprising a biodegradable ester linkage and / or a bioreducible disulfide linkage;

[0103] R’ is hydrophilic sidechain comprising a monovalent radical derived from a hydrophilic amine monomer;

[0104] R” is monovalent radical derived from an amine-containing end capping group; and

[0105] pharmaceutically acceptable salts thereof.

[0106] Further, WO2022067249 disclosed branched PBAEs of the following generic formula:

[0107] ;

[0113] ;

[0114] ;

[0115] ; ;); oftedO N

[0124] O O(B13); the group consisting of 1, 2, 3, 4, monomer is selected(B12); and OOH(S4);OH(S6);OH from: O N (S91).NH2 (E1); NH2 (E2);;OH

[0134] ;

[0136]

[0137] ; N N

[0138] NH2(E63).

[0139] In more particular embodiments, the branched PBAE is selected from 9,8-91-39; 9,12-91- comprises 9,8-91- and siRNA. acid weight-to- 10:1, 11:1, 12:1,13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, and 40:1.

[0142] In certain embodiments, the nanoparticle has a percent branching having a range from about 1% to about 60%, including about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60%.

[0143] In certain embodiments, the nanoparticle has an acrylate:amine (Ac:Am) ratio of 2.2:1 or 2.4:1.

[0144] In certain embodiments, the nanoparticle has a size between about 10 nm to about 150 nm in diameter, including about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, and 150. In particular embodiments, the nanoparticle has a size of about 50 nm.

[0145] In other embodiments, the presently disclosed subject matter provides a method for treating a disease, condition, or disorder, the method comprising administering a therapeutically effective amount of a nanoparticle disclosed herein to a subject in need of treatment thereof. In particular embodiments, the disease, condition, or disorder is selected from a cancer, an autoimmune disease, an ocular disease, and a genetic disease or disorder. In more particular embodiments, the cancer comprises melanoma or hepatocellular carcinoma.

[0146] In other embodiments, the presently disclosed subject matter provides a method for transfecting one or more cells, the method comprising contacting the one or more cells with a nanoparticle disclosed herein.

[0147] As used herein, the term “treating” can include reversing, alleviating, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed compounds can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition.

[0148] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to,primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0149] In general, a “therapeutically effective amount” of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, and the like. In some embodiments, the term “therapeutically effective amount” refers to an amount sufficient to reduce or ameliorate the severity, duration, progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurrence, development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0150] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly a compound disclosed herein and at least one other therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state.

[0151] Further, the compounds disclosed herein can be administered alone or in combination with adjuvants that enhance stability of the compounds, alone or in combination with one or more therapeutic agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0152] The timing of administration of a compound disclosed herein and at least one additional therapeutic agent can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of a compound described herein and at least one additional therapeutic agent either simultaneously, sequentially, or a combination thereof. Therefore, a subject administered a combination of a compound described herein and at least one additional therapeutic agent can receive a compound and at least one additional therapeutic agent at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject.

[0153] When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the compound described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either a compound or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.

[0154] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.

[0155] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” andderivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.

[0156] Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by:

[0157] Qa / QA + Qb / QB = Synergy Index (SI)

[0158] wherein:

[0159] QAis the concentration of a component A, acting alone, which produced an end point in relation to component A;

[0160] Qa is the concentration of component A, in a mixture, which produced an end point;

[0161] QB is the concentration of a component B, acting alone, which produced an end point in relation to component B; and

[0162] Qb is the concentration of component B, in a mixture, which produced an end point.

[0163] Generally, when the sum of Qa / QA and Qb / QB is greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effective amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.

[0164] Depending on the specific conditions being treated, the “agent(s)” may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed- or sustained-slow release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular,intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery.

[0165] For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0166] Use of pharmaceutically acceptable inert carriers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject (e.g., patient) to be treated.

[0167] For nasal or inhalation delivery, the agents of the disclosure also may be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances, such as saline; preservatives, such as benzyl alcohol; absorption promoters; and fluorocarbons.

[0168] In particular embodiments, the compound disclosed herein is administered intranasally in a form selected from the group consisting of a nasal spray, a nasal drop, a powder, a granule, a cachet, a tablet, an aerosol, a paste, a cream, a gel, an ointment, a salve, a foam, a paste, a lotion, a cream, an oil suspension, an emulsion, a solution, a patch, and a stick. As used herein, the term administrating via an "intranasal route" refers to administering by way of the nasal structures.

[0169] Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, the compounds according to the disclosure are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mgper day, and from 5 to 40 mg per day are examples of dosages that may be used. A non-limiting dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, the bioavailability of the compound(s), the adsorption, distribution, metabolism, and excretion (ADME) toxicity of the compound(s), and the preference and experience of the attending physician.

[0170] In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

[0171] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC), and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0172] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0173] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended insuitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.

[0174] Further, one of ordinary skill in the art will recognize that the presently disclosed compounds, and pharmaceutical compositions thereof, include pharmaceutically acceptable salts. Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and include salts of active compounds that can be prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein. The parent form of the compound can differ from the various salt forms in certain physical properties, such as solubility, and the like.

[0175] When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent or by ion exchange, whereby one basic counterion (base) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, magnesium, and the like.

[0176] When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent or by ion exchange, whereby one acidic counterion (acid) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids, organic acids, and amino acids. See, for example, Berge et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Compounds containing both basic and acidic functionalities allow such compounds to be converted into either base or acid addition salts.

[0177] Accordingly, pharmaceutically acceptable salts suitable for use with the presently disclosed subject matter include, by way of example but not limitation, acetate, arginate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, monohydrogencarbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, galactonate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydriodic, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, isobutyrate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate, phthalate, diphosphate,monohydrogen phosphate, dihydrogen phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, suberate, succinate, sulfate, monohydrogensulfate, tannate, tartrate, including (+)-tartrates, (-)-tartrates, and mixtures thereof including racemic mixtures, teoclate, p- toluenesulfonate and trifluoroacetate. Other pharmaceutically acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).

[0178] Unless otherwise noted, the chemical definitions provided immediately herein below are intended to comply with IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997).

[0179] The term “hydrocarbon” as used herein, refers to any chemical group comprising hydrogen and carbon. A hydrocarbon group may be substituted or unsubstituted. As would be known to one of ordinary skill in the art, all valencies must be satisfied in making any substitutions. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.

[0180] The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocyclyl”, “cycloaliphatic”, or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocyclyl” or “cycloalkyl”) refers to a monocyclic C3-C7 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0181] The term “alkane” refers to acyclic branched or unbranched hydrocarbons having the general formula CnH2n+2, and therefore consisting entirely of hydrogen atoms and saturated carbon atoms.

[0182] The term “alkyl” refers to a univalent group derived from an alkane by removal of a hydrogen atom from any carbon atom and having the chemical formula of -CnH2n+1. The groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups H(CH2)n. The groups RCH2, R2CH (R ≠ H), and R3C (R ≠ H) are primary, secondary and tertiary alkyl groups, respectively.

[0183] An alkyl can be a straightchain (i.e., unbranched) or branched acyclic hydrocarbon having the number of carbon atoms designated (i.e., C1-10means one to ten carbons, including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbons). In particular embodiments, the term “alkyl” refers to C1-20inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons. In other embodiments, the alkyl can be a C1-C4 alkyl, including 1, 2, 3, and 4 carbons. In yet other embodiments, the alkyl can be a C1-C6alkyl, including 1, 2, 3, 4, 5, and 6 carbons. In even yet other embodiments, the alkyl can be a C1-C8 alkyl, including 1, 2, 3, 4, 5, 6, 7, and 8 carbons.

[0184] “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, “alkyl” refers to straight-chain alkyls. In other embodiments, “alkyl” refers to branched alkyls. In certain other embodiments, “alkyl” refers to straight-chain and / or branched alkyls. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.

[0185] Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n- hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

[0186] Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more substituents, which can be the same or different. Such substituent groups include, but are not limited to, alkyl, substituted alkyl, cycloalkyl, halogen, acyl, carboxyl, oxo, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, cyano, and mercapto.

[0187] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain having from 1 to 20 carbon atoms orheteroatoms consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, - CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, - CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0188] The term “cycloalkane” refers to saturated monocyclic hydrocarbons (with or without side chains), e.g., cyclobutane. Unsaturated monocyclic hydrocarbons having one endocyclic double or one triple bond are called cycloalkenes and cycloalkynes, respectively. Those having more than one such multiple bond are cycloalkadienes, cycloalkatrienes, and the like. The inclusive terms for any cyclic hydrocarbons having any number of such multiple bonds are cyclic olefins or cyclic acetylenes.

[0189] The term “cycloalkyl” refer to a univalent group derived from a cycloalkane by removal of a hydrogen atom from a ring carbon atom. Cycloalkyls can be a mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group also can be optionally substituted with a substituent group provided hereinabove for alkyl groups. Representative monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl, and fused ring systems, such as dihydro- and tetrahydronaphthalene, and the like.

[0190] The term “cycloalkylalkyl” as used herein, refers to a cycloalkyl group, which is attached to the parent molecular moiety through an alkylene moiety, also as defined above, e.g., a C1-20 alkylene moiety. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

[0191] The terms “cycloheteroalkyl” and “heterocycloalkyl” (or more generally “heterocyclic”) are used interchangeably and refer to an unsaturated ring system, such as a 3- to 10-member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of nitrogen (N), oxygen (O),sulfur (S), phosphorus (P), and silicon (Si), in which the nitrogen, sulfur, and phosphorus heteroatoms may be oxidized and the nitrogen heteroatom may be quaternized. The cycloheteroalkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and / or non-aromatic hydrocarbon rings. Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.

[0192] The terms “cycloalkylene” and “heterocycloalkylene” refer to the divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.

[0193] As used herein the terms “bicycloalkyl” and “bicycloheteroalkyl” refer to two cycloalkyl or cycloheteroalkyl groups that are bound to one another. Non-limiting examples include bicyclohexane and bipiperidine.

[0194] An “unsaturated hydrocarbon” has one or more double bonds or triple bonds. As used herein, the term “alkene” refers to an acyclic branched or unbranched hydrocarbons having one carbon–carbon double bond and the general formula CnH2n. Acyclic branched or unbranched hydrocarbons having more than one double bond are alkadienes, alkatrienes, and the like.

[0195] More particularly, the term “alkenyl” as used herein refers to a monovalent group derived from a C2-20 inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen molecule. Alkenyl groups include, but are not limited to, ethenyl (i.e., vinyl), 2-propenyl, butenyl, 1-methyl-2-buten-1-yl, pentenyl, 2- isopentenyl, hexenyl, octenyl, allenyl, butadienyl, crotyl (but-2-en-1-yl), 2-(butadienyl), 2,4- pentadienyl, 3-(l,4-pentadienyl), and the like, including higher homologs and isomers.

[0196] The term “cycloalkenyl” as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.

[0197] The term “alkyne” as used herein refers to an acyclic branched or unbranched hydrocarbons having a carbon-carbon triple bond and the general formula CnH2n-2, RC≡CR. Acyclic branched or unbranched hydrocarbons having more than one triple bond are known as alkadiynes, alkatriynes, and the like.

[0198] The term “alkynyl” as used herein refers to a monovalent group derived from a straight or branched C2-20 hydrocarbon of a designed number of carbon atoms containing at least one carbon- carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, and heptynyl groups, and the like.

[0199] As used herein, the term “alkylene” refers to an alkanediyl group having the free valencies on adjacent carbon atoms, e.g. –CH(CH3)CH2– propylene (systematically called propane-1,2- diyl). More particularly, the term “alkylene” by itself or a part of another substituent refers to a straight or branched bivalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and / or substituted with one or more “alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (–CH2– ); ethylene (–CH2–CH2–); propylene (–(CH2)3–); cyclohexylene (–C6H10–); –CH=CH–CH=CH–; –CH=CH–CH2–; -CH2CH2CH2CH2-, -CH2CH=CHCH2-, -CH2CsCCH2-, - CH2CH2CH(CH2CH2CH3)CH2-, -(CH2)q-N(R)-(CH2)r–, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (–O–CH2–O–); and ethylenedioxyl (-O-(CH2)2–O–). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being some embodiments of the present disclosure. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

[0200] The term “heteroalkylene” by itself or as part of another substituent means a divalent group derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms also can occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formulaof the linking group is written. For example, the formula -C(O)OR’- represents both -C(O)OR’- and –R’OC(O)-.

[0201] The term “arene” refers to a monocyclic and polycyclic aromatic hydrocarbon.

[0202] The term “aryl” refers to a group derived from arenes by removal of a hydrogen atom from a ring carbon atom. Groups similarly derived from heteroarenes are sometimes subsumed in this definition. An aryl group can include, for example, a single ring or multiple rings (such as from 2 to 3 rings), which are fused together or linked covalently.

[0203] The term “heteroaryl” refers to a group formed by removing one or more hydroxy groups from oxoacids that have the general structure RkE(=O)l(OH)m (l ≠ 0), and replacement analogues of such acyl groups. In organic chemistry an unspecified acyl group is commonly a carboxylic acyl group.

[0204] The term “heteroaryl” refers to the class of heterocyclyl groups derived from heteroarenes by removal of a hydrogen atom from any ring atom. A “heteroaryl” group can include from one to four heteroatoms (in each separate ring in the case of multiple rings) selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2- imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3- isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2- thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The terms “arylene” and “heteroarylene” refer to the divalent forms of aryl and heteroaryl, respectively.

[0205] For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the terms “arylalkyl” and “heteroarylalkyl” are meant to include those groups in which an aryl or heteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like). However, the term “haloaryl,” as used herein is meant to cover only aryls substituted with one or more halogens.

[0206] Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specific number of members (e.g., “3 to 7 membered”), the term “member” refers to a carbon or heteroatom.

[0207] Each of above terms defined hereinabove (e.g. , “alkyl,” “heteroalkyl,” “cycloalkyl, and “heterocycloalkyl”, “alkenyl”, “alkynyl,” “aryl,” “heteroaryl,” as well as their divalent derivatives) are meant to include both substituted and unsubstituted forms of the indicated group. Optional substituents for each type of group are provided below.

[0208] As used herein, the term “acyl” refers to a group formed by removing one or more hydroxy groups from oxoacids that have the general structure RkE(=O)l(OH)m(l ≠ 0), and replacement analogues of such acyl groups. In organic chemistry an unspecified acyl group is commonly a carboxylic acyl group. For example, in some embodiments, the term acyl includes an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent and has the general formula RC(=O)-, wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein). As such, the term “acyl” specifically includes arylacyl groups, such as a 2-(furan-2-yl)acetyl)- and a 2-phenylacetyl group. Specific examples of acyl groups include acetyl and benzoyl. Acyl groups also are intended to include amides, -RC(=O)NR’, esters, -RC(=O)OR’, ketones, -RC(=O)R’, and aldehydes, -RC(=O)H.

[0209] The terms “alkoxyl” or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl–O–) or unsaturated (i.e., alkenyl–O– and alkynyl–O–) group attached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are as previously described and can include C1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, n-butoxyl, sec-butoxyl, tert-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and the like.

[0210] The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.

[0211] “Aryloxyl” refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl. The term “aryloxyl” as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

[0212] “Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

[0213] “Aralkyloxyl” refers to an aralkyl-O– group wherein the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl, i.e., C6H5-CH2-O-. An aralkyloxyl group can optionally be substituted.

[0214] “Alkoxycarbonyl” refers to an alkyl-O-C(=O)– group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and tert-butyloxycarbonyl.

[0215] “Aryloxycarbonyl” refers to an aryl-O-C(=O)– group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

[0216] “Aralkoxycarbonyl” refers to an aralkyl-O-C(=O)– group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

[0217] The term “acyloxyl” refers to an oxygen-centered radicals consisting of an acyl radical bonded to an oxygen atom, e.g., an acyl-O- group wherein acyl is as previously described.

[0218] The term “amine” refers to a compound formally derived from ammonia by replacing one, two or three hydrogen atoms by hydrocarbyl groups, and having the general structures RNH2 (primary amines), R2NH (secondary amines), R3N (tertiary amines). In some embodiments, the term amino refers to the –NH2 group. More generally, the amino group is -NR'R”, wherein R' and R” are typically selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0219] The terms “acylamino” and “alkylamino” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups, respectively.

[0220] An “aminoalkyl” as used herein refers to an amino group covalently bound to an alkylene linker. More particularly, the terms alkylamino, dialkylamino, and trialkylamino as used herein refer to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom. The term alkylamino refers to a group having the structure –NHR’ wherein R’ is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure –NR’R”, wherein R’ and R” are each independently selected from the group consisting of alkyl groups. The term trialkylamino refers to a group having the structure –NR’R”R”’, wherein R’, R”, and R’” are each independently selectedfrom the group consisting of alkyl groups. Additionally, R’, R”, and / or R’” taken together may optionally be –(CH2)k– where k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, isopropylamino, piperidino, trimethylamino, and propylamino.

[0221] The terms alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl–S–) or unsaturated (i.e., alkenyl–S– and alkynyl–S–) group attached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0222] “Acylamino” refers to an acyl-NH– group wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH– group wherein aroyl is as previously described.

[0223] The term “carbonyl” refers to a compound containing the carbonyl group, -C(=O)-. The term is commonly used in the restricted sense of aldehydes (R-C(=O)H) and ketones, although it actually includes carboxylic acids and derivatives.

[0224] The term “carboxylic acid” refers to an oxoacids having the structure RC(=O)OH. The term is used as a suffix in systematic name formation to denote the –C(=O)OH group including its carbon atom. In some embodiments, the term “carboxyl” refers to the –COOH group. Such groups also are referred to herein as a “carboxylic acid” moiety.

[0225] “Carbamoyl” refers to an amide group of the formula –C(=O)NH2.

[0226] “Alkylcarbamoyl” refers to a R’RN–C(=O)– group wherein one of R and R’ is hydrogen and the other of R and R’ is alkyl and / or substituted alkyl as previously described.

[0227] “Dialkylcarbamoyl” refers to a R’RN–C(=O)– group wherein each of R and R’ is independently alkyl and / or substituted alkyl as previously described.

[0228] The term carbonyldioxyl, as used herein, refers to a carbonate group of the formula -O- C(=O)-OR.

[0229] The term “cyano” refers to the -C≡N group.

[0230] The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0231] The term “hydroxyl” refers to the –OH group.

[0232] The term “hydroxyalkyl” refers to an alkyl group substituted with an –OH group.

[0233] The term “mercapto” refers to the –SH group.

[0234] The term “oxo compound” refers to a compounds containing an oxygen atom, =O, doubly bonded to carbon or another element. The term thus embraces aldehydes, carboxylic acids, ketones, sulfonic acids, amides and esters. Oxo used as an adjective (and thus separated by a space) modifying another class of compound, as in oxo carboxylic acids, indicates the presence of an oxo substituent at any position. To indicate a double-bonded oxygen that is part of a ketonic structure, the term keto is sometimes used as a prefix, but such use has been abandoned by IUPAC for naming specific compounds. A traditional use of keto is for indicating oxidation of CHOH to C=O in a parent compound that contains OH groups, such as carbohydrates, e.g., 3-ketoglucose. In some embodiments, the term “oxo” as used herein means an oxygen atom that is double bonded to a carbon atom or to another element.

[0235] The term “nitro” refers to the –NO2 group.

[0236] The term “thio” refers to replacement of an oxygen by a sulfur, e.g., PhC(=S)NH2, thiobenzamide.

[0237] The term “thiol” refers to a compounds having the structure RSH (R ≠ H), e.g., MeCH2SH ethanethiol. A thiol also is known by the term “mercaptan.”

[0238] The term “thiohydroxyl” or “thiol,” as used herein, refers to a group of the formula –SH.

[0239] The term “sulfate” refers to the –SO4 group.

[0240] The term “sulfide” refers to a compound having the structure RSR (R ≠ H) and also are referred to as “thioethers.”

[0241] The term “sulfone” refers to a compound having the structure, RS(=O)2R (R ≠ H), e.g., C2H5S(=O)2CH3 ethyl methyl sulfone.

[0242] The term “sulfoxide” refers to a compound having the structure R2S=O (R ≠ H), e.g., Ph2S=O diphenyl sulfoxide.

[0243] The term “ureido” refers to a urea group of the formula –NH—CO—NH2.

[0244] One of ordinary skill in the art would recognize that a structure represented generally by, for example, the formula: (R)n

[0245] orfor example, but not limited to a 3-carbon, a 4- and the like, aliphatic and / or aromatic cyclic structure, a partially saturated ring structure, and anunsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and number of R groups is determined by the value of the variable “n,” which is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution. Each R group, if more than one, is substituted on an available carbon of the ring structure rather than on another R group. For example, the structure above where n is 0 to 2 would comprise compound groups including, but not limited to: ring structure indicates that the bond can be line representing a bond in a cyclic ring from the group consisting of a saturated ringa an unsaturated ring structure.

[0250] The symbol ( ) denotes the point of attachment of a moiety to the remainder of themolecule.

[0251] When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being “absent,” the named atom is replaced by a direct bond.

[0252] Throughout the specification and claims, a given chemical formula or name shall encompass all tautomers, congeners, and optical- and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

[0253] Certain compounds of the present disclosure may possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolutestereochemistry, as (R)-or (S)- or, as D- or L- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those which are known in art to be too unstable to synthesize and / or isolate. The present disclosure is meant to include compounds in racemic, scalemic, and optically pure forms. Optically active (R)- and (S)-, or D- and L-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefenic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

[0254] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

[0255] As used herein, the term “congener” refers to one of two or more substances related to each other by origin, structure, or function.

[0256] The term “enantiomer” refers to one of a pair of molecular entities which are mirror images of each other and non-superposable.

[0257] The term “stereoisomer” refers to an isomer that possess identical constitution, but which differ in the arrangement of their atoms in space.

[0258] The term “racemate” refers to an equimolar mixture of a pair of enantiomers. It does not exhibit optical activity. The chemical name or formula of a racemate is distinguished from those of the enantiomers by the prefix (±)- or rac- (or racem-) or by the symbols RS and SR.

[0259] The term “diastereoisomerism” refers to stereoisomerism other than enantiomerism. Diastereoisomers (or diastereomers) are stereoisomers not related as mirror images. Diastereoisomers are characterized by differences in physical properties, and by some differences in chemical behavior towards achiral as well as chiral reagents.

[0260] It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure. The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

[0261] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example,compounds having the present structures with the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by13C- or14C-enriched carbon are within the scope of this disclosure.

[0262] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

[0263] The term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / -15%, + / -10%, + / -5%, + / -4%, + / -3%, + / - 2%, and + / -1%. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0264] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0265] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.e., “one or more,” unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be non- limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.EXAMPLES

[0266] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

[0267] EXAMPLE 1

[0268] Efficient Polymeric Nanoparticle Gene Delivery Enabled Via Tri- and Tetrafunctional Branching

[0269] Overview

[0270] Poly( / 3-amino ester) (PBAE) nanoparticles (NPs) show great promise for nonviral gene delivery. Recent studies suggest branched PBAEs (BPBAEs) offer advantages over linear counterparts, but the effect of polymer structure has not been well investigated across many chemical constituents. In this Example, a library of BPBAEs was synthesized with tri- and tetrafunctional branching. These polymers self-assemble with DNA to form highly cationic, monodisperse NPs with notably small size (approximately 50 nm). Optimal transfection occurred with polymer structures that featured moderate PBAE branching, enabling complete DNA encapsulation, rapid NP uptake, and robust expression at low DNA doses and polymer amounts. Optimized NPs enabled efficient DNA delivery to diverse cell types in vitro while maintaining high cellular viability, demonstrating significant improvements over a well-performing linear PBAE counterpart. BPBAEs also facilitated efficient mRNA and siRNA delivery, highlighting the versatility of these structures and demonstrating the broad utility of BPBAE NPs as vectors for nucleic acid delivery.

[0271] Background

[0272] Gene therapy continues to gain increased prominence in medicine, with nucleic acid-based therapeutics reaching the clinic and targeting a wide array of disease areas, Arabi et al., 2022, yet effective and safe nucleic acid delivery remains a challenge for many gene-based therapeutics. Nucleic acid-loaded polymeric nano-particles (NPs) carry several advantages over traditional viralvectors, including low immunogenicity, ease in manufacturability, and large cargo capacities. Nayerossadat et al., 2012. While viral vectors and lipid NPs have advanced to the clinic, Mendell et al., 2017; Wang et al., 2024; Thi et al., 2021; Baden et al., 2021, they are limited in application, however, and have been found in some cases to result in serious adverse events in patients; Kachanov et al., 2024; Maurya et al., 2022; Lee et al., 2023, on the other hand, polymeric gene delivery has thus far lagged behind these more traditional technologies in entering the clinic.

[0273] Poly( / 3-amino ester) (PBAE) NPs are a leading polymeric vector of interest for eventual translation given their high transfection efficacy, hydrolytic degradability, and low cytotoxicity. Sunshine et al., 2012; Wei et al., 2023. Further, PBAEs are manufactured using commercially available reagents and simple chemical reactions, making their production scalable and relatively inexpensive. Karlsson et al., 2020. Subtle changes to polymer structure and NP formulation can vastly affect transfection efficacy, especially among cell types, Sunshine et al., 2012; Bhise et al., 2010, making the design of a formulation with broad applicability challenging.

[0274] Consequently, a highly versatile PBAE NP formulation remains elusive, often necessitating extensive polymer screening and NP optimization with each therapeutic application. Additionally, linear PBAE NPs often require high polymer to nucleic acid weight-to-weight (w / w) ratios for successful transfection which increases cost, may lead to solubility limitations, and may also increase toxicity. Thus, there is a need to develop improved polymer structures that can deliver nucleic acids with lower polymer doses. To overcome these challenges, recent efforts have led to the development of branched PBAEs (BPBAEs), whose complex 3D structures can give rise to favorable NP properties and improved transfection efficacy. Wilson et al., 2019; Zhou et al., 2016.

[0275] Linear PBAEs are synthesized via Michael addition of a diacrylate backbone monomer and amine side chain, leading to a linear base polymer structure. Often, a subsequent Michael addition is performed with an additional amine end-capping monomer, a key mediator of transfection efficacy. Sunshine et al., 2012; Bhise et al., 2010. BPBAEs of varying branching ratios can be generated by the incorporation of multi-acrylate backbone monomers into the base polymer reaction mixture, whereby the ratio of di- and multi-acrylate backbone monomers modulates the degree of polymer branching.

[0276] BPBAEs have been previously synthesized using triacrylate backbone monomers, specifically, trimethylolpropane triacrylate (referred to here as B8), allowing trifunctionalbranching. These structures have enabled successful intracellular delivery of various cargoes including protein, Liu et al., 2022, plasmid DNA, Wilson et al., 2019, minicircle DNA, Zeng et al., 2019; Liu et al., 2019, and siRNA, Wang et al., 2020, as well as DNA / siRNA and DNA / sgRNA in combination. Rui et al., 2019. Further, B8-based BPBAEs have shown promise for several diverse in vivo applications, including therapeutic protein delivery to cancer, Lu et al., 2022, siRNA delivery to the myocardium, Wang et al., 2020, and treatment of genetic skin disease. Cutlar et al., 2016. More recently, pentaerythritol tetraacrylate (referred to here as B12) has been used in select studies as a branching acrylate monomer. Li et al., 2024; Li et al., 2023; Li et al., 2022; Wang et al., 2024; Li et al., 2023. Despite these advances, no direct comparison between BPBAEs synthesized with different branching acrylate monomers has been performed, and the effect of this differential chemistry on NP biophysical properties and transfection performance across cell types is unknown.

[0277] In this Example, we synthesized BPBAEs using B8, B12, and di(trimethylolpropane) tetraacrylate (referred to here as B13), allowing comparison of BPBAEs synthesized with tri- and tetrafunctional branching (FIG. 1). We explored various properties, including the degree of BPBAE branching, acrylate:amine feed ratio, and NP w / w. The optimized BPBAE NPs enabled highly efficient transfection with low nucleic acid dose, w / w, and cytotoxicity. Following characterization of these PBAE structures and resulting NPs, we further demonstrate the versatility of these optimized formulations through successful delivery of various nucleic acid cargoes to diverse cell types.

[0278] Materials and Methods

[0279] Polymer Synthesis

[0280] Monomers used in syntheses included diacrylate backbones B7 (bisphenol A glycerolate (1 glycerol / phenol) diacrylate, Sigma-Aldrich) and B9 (bisphenol A ethoxylate diacrylate, Sigma- Aldrich), triacrylate backbone B8 (trimethylolpropane triacrylate, Alfa Aesar), tetraacrylate backbones B12 (pentaerythritol tetraacrylate, Sigma-Aldrich) and B13 (di(trimethylolpropane) tetraacrylate, Sigma-Aldrich), side chains S4 (4-amino-1-butanol, TCI), S90 (4-(2- aminoethyl)morpholine, Acros Organics), and S91 (3-morpholinopropylamine, Sigma-Aldrich), and end-capping monomers E1 (1,3-diaminopropane, Sigma-Aldrich), E6 (2-(3- aminopropylamino) ethanol, Fluka), E39 (1-(2-aminoethyl)-piperazine, Sigma-Aldrich), and E63 (diethylenetriamine, Sigma-Aldrich). Backbone and side chain monomers were diluted inanhydrous dimethylformamide (DMF) to 200 mg / mL and combined and stirred at 85 °C for 48 h. Where noted, “% branching” refers to the percentage of acrylate functional groups in the monomer feed contributed by either a triacrylate (B8) or a tetraacrylate (B12 or B13) backbone monomer (Table 1), with the remaining acrylates contributed by the diacrylate monomer (B7 or B9). Next, the desired end-cap was diluted in anhydrous tetrahydrofuran (THF) and added to the reaction vial, with a final polymer concentration of 100 mg / mL and end-cap monomer concentration of 0.3 M.

[0281] Table 1. Mol Ratio of Monomer Feed Multiacrylate / diacrylate mol ratio Triacrylate BPBAE Tetraacrylate BPBAE 0 0.00:1.00 0.00:1.00 10 0.07:1.00 0.06:1.00

[0282] The . The resulting polymers w for 24 h under vacuum bef er stocks were stored at -2 beled in terms of their mo, hain monomer- end-cap monomer’. For example, a polymer synthesized with B9 (diacrylate), B8 (triacrylate), S91, and E39 at 50% branching is labeled as 9,8-91-3950%.

[0283] NP Formulation

[0284] NPs were formed by diluting nucleic acid and PBAE to 25-60 eg / mL and 0.6-3.0 mg / mL, respectively, in 25 mM sodium acetate buffer (pH 5.2), with diluted PBAE concentration adjusted based on desired NP w / w. Dilute nucleic acid and dilute PBAE were then combined at a 1:1 volumetric ratio with vigorous mixing, incubated at room temperature for several minutes to allow NP complexation, and used immediately.

[0285] Cell Culture

[0286] B16F10 murine melanoma cells (gifted from Dr. Jonathan Schneck), B16F10 GFP+ cells (generated as described previously, Eltoukhy et al., 2012, using Addgene plasmid #115665; n2t.net / addgene:115665; RRID:Addgene_115665), ARPE-19 human retinal pigment epithelial cells (ATCC), A375 human melanoma cells (ATCC), Hepa1-6 murine hepatocellular carcinoma cells (ATCC), and NIH / 3T3 murine embryonic fibroblast cells (ATCC) were cultured at 37 °C, 5% CO2, in a humidified environment. B16F10 and A375 cells were cultured in RPMI 1640 (Gibco, Thermo Scientific), ARPE-19 cells in DMEM / F12 (Gibco, Thermo Scientific), and Hepa1-6 and NIH / 3T3 cells in DMEM (Gibco, Thermo Scientific), all supplemented with 10% FBS (Sigma) and 1% penicillin-streptomycin (Gibco, Thermo Scientific). For in vitro transfection, cells were seeded at a density of 5,000 cells / well (B16F10, A375, Hepa1-6) or 10,000 cells / well (ARPE-19, NIH / 3T3) in a 96-well plate and allowed to adhere overnight prior to transfection.

[0287] In Vitro Transfection

[0288] For DNA NP transfections, NPs delivering pEGFP-N1 plasmid (Clontech) were formed as described above, then diluted 9-fold in media with supplements before administration to the cell plate, for a final dose ranging from 200-250 ng DNA / well (unless otherwise noted) and w / w ranging from 10-50. After 2 h, all volume was removed from the plate and replaced with 100 eL fresh media. Cells were collected 48 h after NP dosing.

[0289] For mRNA and siRNA NP transfections, NPs delivering GFP mRNA (TriLink Biotechnologies) or Silencer GFP siRNA (Invitrogen) were formed as described above, then diluted 9-fold in media with supplements before administration to the cell plate, for a final dose of 200 ng mRNA or siRNA / well at 20 w / w. Cells were collected 24 h after NP dosing.

[0290] For all transfections, transfection efficacy (GFP expression) was assessed via flow cytometry on an Attune NxT flow cytometer (ThermoFisher) and analyzed in FlowJo (v10.9.0) (FIG. 9A, FIG. 9B. Cell viability (relative metabolic activity) was assessed via CellTiter-Glo luminescence assay (Promega). All in vitro transfection assays were run with n = 4 biological replicates. 20× images were obtained using a fluorescence microscope (Axio Observer, Zeiss).

[0291] Polymer Characterization

[0292] PBAEs were diluted to 10 mg / mL in DMF and filtered, and gel permeation chromatography (GPC) was performed to evaluate PBAE molecular weight (MW, relative to linear polystyrene standards) and polydispersity (PD). GPC measurements (1260 Infinity II withrefractive index detector, Agilent) utilized a flow rate of 2 mL / min (10 mM lithium bromide in DMF) and run time of 18 min / sample eluted at 40 °C through three Polargel-M 300 mm × 7.5 mm columns (PL1117-6800, Agilent) in series. To confirm PBAE structure, polymers were precipitated and washed twice in anhydrous diethyl ether, then dried for 1 h under vacuum before resuspension in chloroform-d. Structure was confirmed via1H NMR (JEOL, 500 MHz).

[0293] NP Characterization

[0294] To evaluate DNA binding / encapsulation efficiency, pEGFP-N1-loaded NPs were diluted 1:1.5 in 25 mM sodium acetate buffer, then diluted 1:1.2 with 30% glycerol (final 5% glycerol). Naked plasmid was diluted in water and 30% glycerol at an equivalent DNA and glycerol concentration to prepared NPs. Prepared NPs and naked DNA were stained with a loading dye, loaded into a 1% gel containing 10 mg / mL ethidium bromide, and subjected to gel electrophoresis. Bands were visualized on a BioDoc-IT imaging system.

[0295] NP zeta potential was assessed via electrophoretic mobility, and NP size (Z-average hydrodynamic diameter) and polydispersity index (PDI) were determined via dynamic light scattering (DLS) using a Malvern Zetasizer Pro (Malvern Panalytical). NPs were formed as described above and diluted 1:4 in 10% PBS (Gibco) before measurements. Samples represent n = 4 independently prepared replicates.

[0296] Number-average NP diameter was assessed via nanoparticle tracking analysis (NTA, NanoSight NS300, Malvern Panalytical). NPs were formed as described above and diluted 1:10,000 in 10% PBS. n = 3 independently prepared NP replicates are shown, with each replicate representing the average of five independent video frames. Calculation of the average number of plasmids per particle was performed as described previously, using the data from the gel electrophoresis study that validated that there was no unencapsulated free DNA present and the data from the NTA analysis that provided the NP concentration for each NP size. Bhise et al., 2012.

[0297] Dye-accessible DNA in the NPs was assessed using a heparin-competition DNA release assay. NPs were diluted 1:18.75 with 25 mM sodium acetate buffer and plated into a clear-bottom black assay plate. A standard curve of naked DNA was also prepared in 25 mM sodium acetate buffer. An equal volume of 1:200 dilute RiboGreen dye (Invitrogen) in PBS was added to each well and incubated at room temperature for 5 min. Heparin (Sigma-Aldrich) was diluted in PBS and added to the NPs for final concentrations ranging from 0.0-150.0 eg / mL and incubated at roomtemperature for 1 h. Fluorescence was assessed via plate reader (Varioskan LUX, ThermoFisher). All conditions were run in triplicate.

[0298] DNA Uptake Assay

[0299] pEGFP-N1 DNA was labeled with Cy5 using a Label IT Nucleic Acid Labeling Kit (Mirus Bio) according to manufacturer’s instructions. NPs were complexed using the resulting Cy5- pEGFP-N1 DNA and dosed to B16F10 cells as described above. 30, 60, and 120 min later, media and NPs were removed, and the cells were washed twice with 50 eg / mL heparin in PBS to remove residual NPs and halt further internalization, then cells were collected for analysis via flow cytometry (FIG. 9C, FIG. 9D). Each condition was run in quadruplicate.

[0300] Statistical Analysis

[0301] All statistical tests were performed using GraphPad Prism 10. Where noted, a one-way or two-way ANOVA with Dunnett’s posttest comparing all conditions to PBAE 7,8-4-6, the linear polymer 9-91-39, or untreated (UT) control was performed. For the heparin competition release study, a best-fit sigmoidal curve was plotted and an EC50 was determined. Significance is denoted: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

[0302] Results and Discussion

[0303] B8-based BPBAE Synthesis and Evaluation

[0304] We first aimed to synthesize a library of diverse BPBAE structures. BPBAEs were synthesized using diacrylates B7 or B9, side chains S4, S90, or S91, and end-caps E1, E6, E39, or E63 (FIG. 2A). All polymers were synthesized using B8 at 50% branching and an acrylate:amine (Ac:Am) ratio of 2.2:1, resulting in a starting library of 15 distinct structures. For screening purposes, two diverse cell types were selected: B16F10 murine melanoma and ARPE-19 human retinal pigment epithelial cells.

[0305] Despite large differences in transfection efficacy across BPBAE structures, similar trends in transfection were observed between the two cell types (FIG. 2B, FIG. 2C). 9,8-91-39 demonstrated the greatest transfection efficacy across both cell types, and showed 7.7- and 2.1- fold higher GFP mean fluorescence intensity (MFI) in B16F10 and ARPE19 cells, respectively, compared to 7,8-4-6, a previously reported BPBAE structure. Wilson et al., 2019. Importantly, all tested polymers retained cell viability over 80% (FIG. 2D). Based on these screening results, B9, S91, and E39 were used in all subsequent syntheses.

[0306] Synthesis and Assessment of BPBAEs Utilizing Various Branching Monomers

[0307] Based on the high levels of transfection seen with 9,8-91-39 (FIG.2), we next synthesized a library of BPBAEs: 9,8-91-39, 9,12-91-39, and 9,13-91-39 (FIG. 3A) at 0 (linear equivalent, 9- 91-39), 10, 20, 30, 40, 50, and 60% branching and with Ac:Am ratios of 2.2:1 and 2.4:1. B8, which is a triacrylate monomer, generated BPBAEs with trifunctional branching, while tetraacrylate monomers B12 and B13 generated BPBAEs with tetrafunctional branching. Increased MW and PD were observed with increased branching, a trend observed across all three branching monomers (FIG. 2, Table 2).

[0308] Table 2. Relative MW of Linear (0%), Moderately Branched (30%), and Highly Branched (60%) 2.4:1 Ac:Am PBAEs as Determined via GPC branching % branching Mn(Da) Mw(Da) PD monomer N / A 0 1752 5262 3.00 B8 30 1941 7809 4.02 -91- tely Mn, MR FIG.

[0310] We next assessed the effects of branching percentage and Ac:Am feed ratio on BPBAE NP transfection efficacy. B16F10 cells were transfected with GFP DNA-loaded NPs at 25 and 50 w / w delivering 200 ng DNA per well. BPBAEs synthesized at a 2.4:1 Ac:Am before end-capping slightly outperformed 2.2:1 Ac:Am BPBAEs, though transfection results indicated relativelyconsistent transfection efficacy with 2.2:1 and 2.4:1 Ac:Am BPBAEs (FIG. 3B). Interestingly, at 25 w / w, a notable increase in transfection was observed between 0 and 30% branching, followed by decreasing transfection between 30 and 60%, in both 2.2:1 and 2.4:1 Ac:Am polymers, though this trend was less pronounced with 50 w / w NPs (FIG. 3B). This observation suggests that branching percentage and NP w / w may work in tandem, where lower transfection efficiency with suboptimal branching percentage can be rescued by a higher polymer dose.2.4:1 Ac:Am BPBAEs were used in subsequent experimentation.

[0311] To further investigate how nanoparticle w / w would affect transfection efficacy, we transfected B16F10 cells with NPs at 10, 20, and 40 w / w. Poor transfection was observed at 10 w / w regardless of branching percent or branching monomer, and robust transfection was observed at all degrees of branching at 40 w / w (FIG. 4A). Interestingly, when w / w was decreased from 40 to 20, low (less than 30%) and highly branched (greater than 40%) NPs led to lower transfection efficacy, while moderately (30%) branched NPs led to equal or slightly higher transfection efficacy (FIG. 4A). At 20 w / w, 9,12-91-39 30% and 9,13-91-39 30% NPs outperformed their 40 w / w counterparts, suggesting that, while 40 w / w was required for effective transfection for low and highly branched BPBAEs, moderate branching led to greater delivery efficiency and allowed for a lower w / w to be used. Of note, 20 w / w NPs demonstrated peak transfection efficacy in the 30- 40% branching range regardless of branching monomer (FIG. 4A), and high levels of GFP expression were confirmed via fluorescence microscopy (FIG. 4B). This finding was interesting given the structural diversity among the branching monomers and suggests that the ability to most efficiently deliver DNA with approximately 30% branching is an intrinsic property of the BPBAEs and is not specific to the branching monomer.

[0312] Despite slight differences in maximal transfection efficacy (20 w / w NPs at 30% branching), all three BPBAE series show similar trends in transfection efficacy between 10, 20, and 40 w / w NPs: poor transfection at 10 w / w, relatively consistent transfection at 40 w / w, and moderate (30-40%) branching enabling high efficacy at 20 w / w (FIG. 4A). This result suggests there is a critical w / w threshold whereby moderate branching improves transfection efficacy and allows minimized w / w, however, branching becomes less advantageous at higher w / w. When assessing the effects of linear, moderate (30%), or high (60%) branching on cell viability, only 9,8-91-39 and 9,13-91-39 NPs at 60% branching and 40 w / w led to viability below 80% (FIG. 4C). This observation highlights the importance of minimizing the w / w needed for efficienttransfection, as this decreases the total polymer dose, reducing toxicity. This also indicates a potential increase in NP toxicity at high branching percentages, further supporting moderately branched PBAEs for optimal transfection. At greater NP dosages (greater than 2 ng / μL DNA), 20 w / w 9,13-91-3960% NPs demonstrated notably increased toxicity compared to linear and 30% branched formulations, while 9,12-91-3960% NPs demonstrated reduced toxicity (FIG.21). This was notable given that 9,13-91-3960% NPs, but not 9,12-91-3960% NPs, were able to achieve transfection at 20 w / w (FIG. 4A). This suggests that branching degree, transfection efficacy, and cytotoxicity are interrelated.

[0313] Eltoukhy et al., 2012, observed a similar switch-like behavior of linear PBAE transfection efficacy, with robust transfection at 40 w / w and minimal transfection at 20 w / w, as observed here (FIG. 4A). Further, they demonstrated linear PBAE transfection efficacy is optimal with Mwin the approximately 5 kDa range, which was seen here by linear 9-91-39 (Table 2). Nonetheless, given the increases in MW observed with increased branching, we sought to determine if increasing the MW of linear PBAE 9-91-39 could recover its transfection efficacy at 20 w / w. Indeed, linear 9-91-39 PBAEs with Mn greater than B8, B12, and B13 moderately branched (30%) PBAEs failed to achieve robust transfection at 20 w / w (Table 3, FIG.22). Further, highly branched (60%) formulations saw decreased transfection at 20 w / w compared to their moderately branched (30%) counterparts (FIG. 4A), despite their increased MW (Table 2). Without wishing to be bound to any one particular theory, it is thought that the advantages in transfection efficacy observed with moderate branching are not dependent on the corresponding increase in MW over their linear counterpart.

[0314] Table 3. GPC characterization of high MW linear 9-91-39 PBAEs. To increase PBAE MW, polymers were synthesized at an Ac:Am of 2.1:1 and monomer concentration of 200 mg / mL or 400 mg / mL prior to endcapping. Polymer % Branching Ac:Am mg / mL Mn (Da) Mw (Da) PD Ratio 9-91-39 0 2.10:1.00 200 2383 4533 1.90 9-91-39 0 2.10:1.00 400 3606 7526 2.09[00315 [00316 and 9,13- 91-39 nd highly branch ar control structulete DNA binding was observed regardless of branching, as demonstrated via gel electrophoresis (FIG. 5B). Surface charge measurements revealed high zeta potential (ZP) in all formulations; all NPs had a ZP greater than +30 mV (FIG. 5C). This high surface charge was accompanied by small NP size, with all formulations having NP diameters at approximately 50 nm as measured via DLS (FIG. 5D). Further, all NP formulations maintained high monodispersity, with PDI values less than 0.2 FIG. 5E). This was consistent across additional NP w / w’s: assessment of 10 and 40 w / w NPs showed markedly similar NP surface charge, size, and PDI, with only B12 60% 40 w / w NPs reaching a PDI slightly greater than 0.2 (FIG. 23). A modest trend in increasing NP PDI was observed with increasing degree of branching, which may be linked to the corresponding increases in PBAE PD observed with increased branching (FIG. 10, Table 2).

[0317] The majority of previously reported BPBAE structures have utilized an amino alcohol side chain and ionizable amine end-cap. As branching increases the theoretical number of end-caps per molecule, Wilson et al., 2019, these polymers have a substantially higher density of ionizable amines when branched than their linear counterparts. Consequently, increased branching in prior BPBAE formulations resulted in increased NP surface charge, decreased NP size, and decreased the required w / w for complete DNA binding. Zhou et al., 2016. In contrast, the BPBAE formula- tions developed here contain a highly ionizable amine moiety (S91, 3-morpholinopropylamine) as their side chain, with an additional ionizable amine end-cap (E39, 1-(2-aminoethyl)- piperazine). Thus, the density of ionizable amines present is substantially greater than that of prior PBAE formulations, Sunshine et al., 2012; Wilson et al., 2019; Zhou et al., 2016; Li et al., 2024; Bishop et al., 2013, which likely relates to the notably high surface charges and low NP sizes observed here. Further, ionizable amines are present in high density throughout the polymer backbone, rather than concentrating in polymer end-caps, leading to more consistent cationicity regardless of branching (FIG. 5C). Furthermore, branching did not significantly affect DNA condensation ability or NP size (FIG. 5B, FIG. 5D). Taken together, these results suggest that NP physical properties are likely dominated by the cationicity of the formulationand are not intrinsic to the branching structure itself. Despite similar NP properties observed across branching percentage and branching monomers at 20 w / w (FIG. 5B-FIG. 5E), however, only 30% branched NPs were able to yield robust transfection (FIG. 4A).

[0318] High cationicity has been shown to be beneficial for BPBAE NP uptake, Li et al., 2024, however, others have hypothesized that excessively high NP cationicity can increase cytotoxicity from disruption of cellular membranes. Karlsson et al., 2020. Incorporation of cationic side chains into BPBAEs was recently explored by Li et al., 2024. Formulations were similarly optimized at 20 w / w to maximize transfection efficacy while minimizing toxicity, and incorporation of cationic side chains enabled 20 w / w NPs with greater than 85% DNA encapsulation, surface charge approaching +30 mV, and NP size 400 nm. In contrast, the exclusive use of a cationic side chain in the BPBAE library used here resulted in increased cationicity (approaching +40 mV), substantially smaller size (50 nm) and near-complete DNA encapsulation at 20 w / w. Li et al., 2024, found the introduction of backbone cationicity decreased BPBAE NP cytotoxicity, which aligns with the findings here that NPs demonstrated minimal toxicity at 20 w / w.

[0319] We then confirmed the size of the NP formulations via NTA (FIG. 5F). 9-91-39 (0% branched) NPs were 52 nm, 30% branched NPs ranged from 53 to 55 nm, and 60% branched NPs from 63 to 66 nm (FIG. 5G), which largely agrees with the results from DLS. This consistency in hydrodynamic diameter measurement between DLS (intensity-averaged) and NTA (number- averaged) further demonstrates that the NPs are monodisperse, without larger aggregates forming. NTA also allowed determination of NP concentration, and, using the assumption of complete DNA binding, as observed in FIG.5B, we then estimated the approximate number of plasmid copies per NP (FIG. 5H, FIG. 5I) based on the encapsulated 4.7 kbp pEGFP-N1 plasmid. Approximate plasmids per NP ranged from 2 (B8 30%) to 4 (B12 60%) (FIG. 5H). Prior work utilizing an equivalently sized plasmid with linear PBAEs formed NPs of significantly larger size and higher loading that ranged from 30 to 120 plasmids per NP, Bhise et al., 2012, indicating that these BPBAEs complex with far fewer plasmid copies per NP compared to prior formulations. Here, the small size and a greater number of NPs formed at a given DNA dose proved highly effective for DNA delivery, and future use of smaller DNA constructs, such as minicircle or nanoplasmid DNA, may further increase transfection efficacy by increasing the number of plasmid copies per NP. While small particle size could improve transport and uptake, a lower loading of plasmids per NP may prove less advantageous for codelivery of multiple plasmids or nucleic acid types, as loadinghigh numbers of plasmid copies per PBAE NP is beneficial for coexpression of multiple delivered constructs. Bhise et al., 2012. Thus, given the small size and consequently low counts of nucleic acid molecules per NP, these BPBAE NPs are potentially less likely to coencapsulate multiple constructs and deliver both simultaneously to a cell.

[0320] We assessed the ability of the BPBAE NPs to shield DNA from a fluorescent DNA intercalating dye when challenged with heparin, a competitive binder to the polymer. All tested formulations successfully shielded greater than 98% of DNA before being challenged. Near total DNA release was observed with sufficient heparin challenge; over 98% of DNA was dye- accessible when NPs were challenged with 27.8 ig heparin / ig DNA (FIG. 5J). Interestingly, the ability of the NPs to exclude dye under heparin challenge clustered by branching percentage. 9- 91-39 (0% branched) NPs were least resistant to heparin challenge with an EC50 (95% CI) of 8.6 (8.0-9.3) ig heparin / ig DNA (Figure 5I). 30% branched NPs were more stable, with B830%, B12 30%, and B1330% NPs having EC50s of 11.8 (10.8-12.7), 11.9 (10.2-13.6), and 12.3 (11.4-13.1) ig heparin / ig DNA, respectively, followed by B860%, B1260%, and B1360% NPs, at 13.9 (11.1- 16.7), 14.0 (13.0-15.0), and 15.1 (13.6-16.6) ig heparin / ig DNA, respectively (FIG. 5J). Together, this indicates that increased branching led to NPs that were more resistant to heparin challenge and that BPBAEs, with a higher density of amine end groups compared to linear structures, bind DNA more tightly, likely due to avidity effects of the extra amines. Bishop et al., 2013; McGhee and von Hippel, 1974. Without wishing to be bound to any one particular theory, it is thought that moderate stability may be most optimal given that 30% branching formulations, which offer moderate protection of DNA when challenged with heparin, also led to the most robust transfection efficacy (FIG. 3B and FIG. 4A), suggesting a required balance of DNA protection and release for optimal intracellular delivery. This biphasic response to DNA-polymer binding affinity has also been reported in the literature with linear polymers. Bishop et al., 2013; Schaffer et al., 2000.

[0321] Assessment of BPBAE NP Uptake Efficiency and Kinetics

[0322] We next assessed whether PBAE branching would contribute to differences in NP uptake kinetics. Administration of Cy5-labeled GFP DNA-loaded NPs to B16F10 cells showed high rates of cell internalization, with all NP formulations except B1260% taken up by greater than 93% of cells 120 min following NP administration (FIG. 6A). The lower rate of internalization with B12 60% NPs may help explain the notably poor transfection efficacy seen with that formulation (FIG. 4A). The overall amount of NP uptake also varied by branching percentage and branchingmonomer identity; after 120 min, only B8 30%, B12 30%, and B13 30% showed significantly higher NP uptake compared to 0% branched NPs, and total uptake of B12 60% NPs was again significantly lower compared to other formulations tested (FIG.6B, FIG.6C). A strong correlation was observed between NP uptake and transfection efficacy (FIG. 6D), as measured by Cy5 MFI at 120 min (FIG. 6B) and GFP MFI at 48 h (FIG. 4A), respectively.

[0323] Optimized BPBAE NPs Successfully Transfect Diverse Cell Types

[0324] We next assessed whether the improvements in DNA transfection efficacy afforded by moderate branching could be extended to multiple cell types, comparing 30% branched NPs to 0% branched NPs. BPBAE NPs led to highly efficient transfection in human and murine cancer and noncancer cells, with significant improvements over 0% branched NPs in ARPE-19 human retinal pigment epithelial cells, A375 human melanoma cells, Hepa1-6 murine hepatocellular carcinoma cells, and NIH / 3T3 murine fibroblasts (FIG.7A-FIG.7D, left, middle, FIG.7E). Consistent trends in transfection efficacy were observed across all cell types, with B1330% enabling the greatest transfection efficacy, followed by B8 30% and B12 30%. Notably, all 30% formulations significantly outperformed linear 0% branched 9-91-39 (P < 0.01 for all except for B1230% GFP MFI in NIH / 3T3 cells) (FIG.7A-FIG.7D, left, middle, FIG.7E). All formulations also maintained cell viability over 80% with the exception of B1330% in A375 and Hepa1-6 cells, which dropped to 73% and 74%, respectively (FIG. 7A-FIG. 7D, right), suggesting the increased transfection efficacy afforded by the B13 branching monomer may come with slightly increased cytotoxicity.

[0325] Here, the top-performing BPBAE NP formulation (B1330%) achieved 58% transfection in ARPE-19 cells while maintaining cell viability of 96.5%. This is comparable to prior work optimizing BPBAEs for ARPE-19 gene delivery, which achieved 77% transfection and low toxicity while using a 3-fold higher DNA dose of 20 w / w NPs. Wilson et al., 2019. In contrast, prior optimized linear PBAE NPs have only reached 44% transfection efficacy in these cells and were substantially less efficient, requiring a 3-fold increase in NP w / w (60 w / w) and 3-fold increase in total DNA dose, amounting to a 9-fold increase in total PBAE dose to achieve this efficacy and resulting in cell viabilities below 80%. Sunshine et al., 2012. Further, the present formulation outperforms commercially available vectors, Wilson et al., 2019, (JetPRIME, Lipofectamine 2000, 4 kDa linear PEI, 25 kDa branched PEI), which have been unable to achieve greater than 50% transfection efficacy in ARPE-19 cells without substantial toxicity. Taken together, these results demonstrate the ability of these BPBAE NPs to efficiently transfect diversecell types with low toxicity, improving efficacy over linear structures and highlighting their broad utility as vectors for DNA delivery in many cellular contexts.

[0326] BPBAE NPs Enable Gene Expression and Knockdown via mRNA and siRNA Delivery

[0327] To further explore the versatility of these NP formulations, we next examined whether the BPBAE NPs could deliver other types of nucleic acid. NPs loaded with mRNA encoding GFP led to robust expression 24 h following NP dosing (FIG. 8A). Additionally, NPs loaded with siRNA against GFP led to robust knockdown in B16F10 cells engineered to stably express GFP (FIG. 8B). Interestingly, while moderate branching improved DNA transfection efficacy regardless of the branching monomer at 20 w / w (FIG. 4A and FIG. 7), these results did not extend to all chemistries for RNA delivery. Branched PBAEs B8 30% and B13 30% NPs significantly outperformed linear (0% branched) PBAE 9-91-39 at both mRNA and siRNA delivery, while B12 30% NPs had marginal benefits (siRNA) or negative effects (mRNA) on transfection (FIG. 8A, FIG. 8B).

[0328] At 20 w / w, 0% branched NPs poorly delivered DNA (FIG. 7A-FIG. 7E) but were able to satisfactorily deliver mRNA (43.3% GFP+) and siRNA (30.2% GFP knockdown) (FIG. 8A, FIG. 8B). Nonetheless, moderate branching still enabled significant increases in RNA transfection efficacy, with B1330% NPs achieving a greater than 3-fold increase in mRNA expression (FIG. 8A, middle) and greater than 2-fold increase siRNA knockdown (FIG. 8B, left) compared to 0% branched NPs. It is possible that RNA delivery requires weaker binding to the PBAE, given that release in the cytosol is required for expression, rather than transport further to the nucleus, and prior studies have found that PBAEs with tight binding to nucleic acid performed better at DNA delivery, while weakly binding PBAEs were more suited for siRNA delivery.30,31Given this, and the results showing greater nucleic acid shielding with increased branching (FIG.5J), it is possible that the advantages of branching are less significant for RNA delivery compared to DNA delivery, or, that the advantages of branching can be achieved at even lower w / w. This warrants further investigation, particularly for applications where DNA / RNA codelivery are of interest, in which case branching may need to be optimized to allow optimal delivery of both cargoes.

[0329] Both mRNA- and siRNA-loaded NPs maintained cell viability greater than 80%, except for siRNA-loaded B1330% NPs, which dropped to 76% (FIG. 8A, FIG. 8B, right) and is similar to the trends in viability observed with DNA-loaded NPs (FIG.7A-FIG.7D, right); B1330% NPs led to the greatest transfection efficacy, but at the cost of slightly increased toxicity. We alsoinvestigated the NP properties of this top-performing formulation, which were comparable to pDNA-loaded NPs (FIG. 24). B13 30% NPs had a surface charge of +40.7 and +37.6 mV for mRNA and siRNA NPs, respectively (FIG. 24A). NPs loaded with siRNA were 55.1 nm as measured via DLS, while mRNA NPs grew slightly to 77.7 nm (FIG. 24B). Finally, mRNA- and siRNA-loaded NPs had a PDI of 0.21 and 0.27, respectively (FIG. 24C).

[0330] Taken together, these results demonstrate that moderate PBAE branching is able to confer significant improvements in transfection efficacy across a diversity of nucleic acid types (DNA / RNA) and MWs (mRNA / siRNA). Further, these NPs demonstrate potential as highly versatile gene transfer vehicles.

[0331] Overall, this work introduces several contributions to the field of BPBAE gene delivery. First, this study utilized a variety of branching monomers, allowing for a deeper understanding of the effects of this chemistry on NP properties and transfection efficacy. Trifunctional and tetrafunctional branching led to significant improvements over a control linear PBAE, demonstrating significant increases in NP uptake (FIG. 6B) and transfection efficacy (FIG. 7 and FIG. 8) without large differences in NP physical properties (FIG. 5). Of note, approximately 30% branching demonstrated optimal transfection for both branching types, a finding that may prove useful to the rational design of future BPBAEs. Both trifunctional, Wilson et al., 2019; Zeng et al., 2019; Liu et al., 2019; Wang et al., 2020; Rui et al., 2019, and tetrafunctional, Li et al., 2024; Li et al., 2023; Li et al., 2022; Wang et al., 2024; Li et al., 2023, branching are being increasingly explored through the use of multifunctional acrylate monomers. PBAE branching has also been enabled by multifunctional amine monomers, Bo et al., 2024; Chu et al., 2024, with a head-to-head comparison finding multifunctional acrylate branching, as used here, to be the more effective of the two methods. Wang et al., 2020.

[0332] We found that PBAE MW and PD both increased with increased branching regardless of branching monomer (Table 2). BPBAE B1260% resulted in notably high PD (Table 2) and greatly underperformed in terms of both NP uptake and transfection efficacy (FIG.6D). Increasing PBAE MW and decreasing PD have been shown to improve transfection efficacy, a process that can be performed in future work through GPC fractionization, Eltoukhy et al., 2012, or successive washes in acetone / diethyl ether mixtures. Li et al., 2023. Increased branching degree has been shown to increase BPBAE Mw, PD, and the mass fraction of end-caps per molecule. Wilson et al., 2019. Our results support these findings. Future work is important to further quantify the differential effectsof various branching structures through further characterization of branching degree and uncoupling of branching degree from increased molecular weight. Interestingly, the changes in MW observed with increased branching did not appear to affect NP size, with all tested formulations resulting in sizes of approximately 50 nm (FIG. 5D). PBAEs have rarely been reported to form stable NPs below 70 nm, with most formulations ranging from 150 to 450 nm. Zhou et al., 2016; Bhise et al., 2012; Li et al., 2024; Bishop et al., 2013.

[0333] Liu et al., 2017, have also described PBAE NPs below 60 nm and found the incorporation of PBAE branching reduced NP size from 60-140 nm to less than 50 nm. Small NP size holds several consequences on the utility of gene-loaded NPs. First, reduction of NP size to less than 200 nm is expected to result in clathrin-mediated, rather than caveolae-mediated, endocytosis. Rejman et al., 2004; Panyam et al., 2002. Given the changes in uptake mechanism in this range, NP size has been proposed as a potential method of cell type targeting. Rejman et al., 2004; Prabha et al., 2016.

[0334] Second, further reductions to less than 100 nm significantly improves cellular uptake of AuNPs, Prabha et al., 2016, PLGA NPs, Prabha et al., 2002, and PBAE NPs, Anderson et al., 2005, with 10-60 nm seen as the optimal NP size for maximal nonphagocytic cellular uptake in vitro regardless of NP composition or surface charge, Hoshyar et al., 2016, a range achieved with the formulations explored here. NP sedimentation significantly affects in vitro transfection and presents an advantage for larger NPs in laboratory studies, with particular advantage seen with aggregated DNA-loaded PEI NPs. Pezzoli et al., 2017; Ogris et al., 1998. This did not appear to affect the overall efficacy demonstrated by the small BPBAE NPs in this Example, however, as nearly 100% of cells internalized NPs within 2 h of NP dosing (FIG. 6A). Third, NPs must generally be less than 200 nm to avoid the complement system, Hoshyar et al., 2016, and less than 100 nm to cross vascular fenestrae and diffuse into tissue. Prabha et al., 2016; Mathew et al., 2020. Thus, the NP sizes achieved here are of high interest for in vivo gene delivery. Taken together, sub-100 nm size has been proposed as a necessary innovation for gene delivery NPs to reach clinical relevance, Prabha et al., 2016, further encouraging additional exploration of the structures developed here. Future work should more rigorously assess methods to modulate BPBAE NP size. For example, PBAE MW and PD, nucleic acid MW, and complexation concentration may all modulate NP size, with prior work demonstrating even the vigor of polymer / nucleic acidmixing effecting polymeric NP size. Pezzoli et al., 2017. Such an investigation could further narrow the NP sizes most amenable to effective and targeted transfection in vitro and in vivo.

[0335] While this Example discloses an ionizable amine side chain, resulting in NPs with surface charges of approximately +40 mV (FIG. 5C), Liu et al., 2017, used an alkylated side chain, resulting in BPBAE NPs with surface charges between +2 and +10 mV. NP cationicity allows interaction with anionic cell membranes and promotes endocytosis, Prabha et al., 2016, but also encourages more rapid macrophage uptake. Gustafson et al., 2015. Further, NP surface charge greatly affects circulation times and protein coronae in vivo. Mitchell et al., 2021. Thus, further investigation on the balance of ionizable and lipophilic side chains within BPBAEs is warranted, as this may present a method to modulate NP surface charge and substantially affect in vivo pharmacokinetics and cell-type specificity.

[0336] Initial BPBAE screening was performed in B16F10 cells (a rapidly growing, traditionally easy to transfect, murine, cancerous cell line) and ARPE-19 cells (a slower growing, traditionally difficult to transfect, human, noncancerous cell line) (FIG. 2). These two cell types were selected given their various differences with the aim to develop a BPBAE formulation with broad transfection ability. Thus, the high correlation in transfection ability across BPBAE formulations between the two cell types was an exciting result, with B9, S91, and E39 leading to optimal transfection in both cell types (FIG. 2C). B16F10 and ARPE-19 cells were also selected given their relevance to two leading in vivo applications of PBAE-mediated transfection: intratumoral, Wu et al., 2018; Tzeng et al., 2020; Luly et al., 2023, and retinal, Sunshine et al., 2012; Shen et al., 2024, gene delivery, respectively. Thus, there is extensive literature detailing the in vitro efficacy of prior linear and branched PBAE formulations in either cell line with which these results can be compared. Wilson et al., 2019; Sunshine et al., 2012; Chu et al., 2024; Tzeng et al., 2020.

[0337] Both B16F10 and ARPE-19 cells are epithelial lines, however, motivating additional experimentation to expand the diversity of cell types tested (FIG. 7). Transfection efficacy scaled with cell type, however, moderate branching improved transfection efficacy over the linear equivalent PBAE at 20 w / w in all tested cell types (FIG.4A, FIG.7). Second, the ability to robustly transfect a diversity of cell types alludes to the development of a NP formulation with properties intrinsic for effective transfection. Finally, this versatility demonstrates the potential for these formulations to enable effective transfection in a multitude of applications.

[0338] Finally, this Example resulted in a BPBAE NP formulation capable of successful transfection of DNA, mRNA, and siRNA at low doses and w / w, a notable exception in a field where formulations must often be optimized to deliver a specific construct of interest. Thus, these NPs represent a promising vector for future investigation of combinatorial gene delivery.

[0339] Summary

[0340] We explored a library of BPBAEs with trifunctional and tetrafunctional branching in equivalent PBAE backbone structures for head-to-head comparisons of nucleic acid delivery to a variety of cell types and with a variety of nucleic acid cargoes. Moderate branching (30%) allowed robust transfection with 9,8-91-39, 9,12-91-39, and 9,13-91-39 BPBAE NPs at 20 w / w, effectively reducing the required polymer dose in half, as the linear equivalent (9-91-39) required 40 w / w for robust DNA transfection. The use of a highly ionizable PBAE side chain led to surface charges approaching +40 mV, allowing efficient DNA encapsulation and particles as low as 50 nm in size that encapsulated 2-4 DNA plasmids per particle. PBAE branching contributed to significant increases in DNA binding and NP uptake compared to the linear control. For delivery of mRNA or siRNA, the optimized branched polymers 9,8-91-39 30% and 9,13-91-39 30% had superior efficacy compared to linear PBAE 9-91-39. This work contributes to a rapidly developing space of BPBAE gene-loaded NPs, and these BPBAE NP formulations present a promising platform for safe, versatile, and highly efficient gene delivery.

[0341] EXAMPLE 2

[0342] Representative 2.4:1 Ac:Am BPBAEs

[0343] Unless noted otherwise, all BPBAEs in this Example are 2.4:1 Ac:Am unless noted otherwise.

[0344] The visualization of representative PBAE formulations is shown in FIG 25. PBAEs were formulated as described herein at 20 w / w and imaged via transmission electron microscopy (TEM) with negative staining. Two representative images are shown per formulation.

[0345] FIG. 26 shows highly efficient BPBAE-mediated mRNA delivery to human retinal pigment epithelial cells in vitro. ARPE-19 cells (10k / well) were transfected with 9,8-91-39 (30%, 2.4:1) or canonical linear PBAE 4-5-7 NPs loaded with GFP mRNA.

[0346] FIG. 27A, FIG. 27B, FIG. 27C, and FIG. 27D show in vitro PBAE screening in Madin- Darby Canine Kidney (MDCK) cells. (FIG. 27A) Monomers tested. (FIG. 27B) Transfection efficacy assessed via fLuc DNA-loaded NPs. (FIG.27C) Cell viability (relative metabolic activity)assessed via GFP DNA-loaded NPs. (FIG. 27D) 10× images (Green = GFP; scale bar = 250 µm) of highest performing condition of top two polymer formulations.

[0347] FIG. 28A, FIG. 28B, and FIG. 28C demonstrate development of nanoplasmid-loaded lyophilized BPBAE NPs. NPs were complexed with nanoplasmid GFP DNA and polymer 9,8-91- 39 at 20 w / w and lyophilized, using sucrose as an excipient. NPs were complexed to have a final DNA concentration after reconstitution in water of 0.033 µg / µL (low dose, LD) or 0.1 µg / µL (high dose, HD) and final sucrose concentration of 100 µg / µL. (FIG.28A) NP size, polydispersity index (PDI), surface charge, and osmolarity following reconstitution. (FIG. 28B) Reconstituted NPs successfully transfect MDCK cells in vitro (10× images, Green = GFP; scale bar = 250 µm, 5k cells / well, %Viability assessed via CTG). (Fig. 28C) Reconstituted NPs successfully transfect ARPE-19 cells in vitro (10k cells / well, transfection assessed via flow cytometry, %Viability assessed via CTG).

[0348] FIG. 29A and FIG. 29B demonstrate the development of BPBAEs synthesized in a single step. (FIG. 29A) B9 and S91 were reacted using the BPBAE backbone reaction previously described here, with Ac:Am ratios of 1.5:1 to 1.9:1 (legend) followed by ether purification and DMSO resuspension as previously described here. This resulted in PBAE 9-91 in a single reaction step. PBAE 9-91 was then complexed with eGFP-N1 plasmid at various w / w (x-axis) and dosed to B16F10 cells in vitro. (FIG. 29B) B9, B8, and S91 were reacted using the BPBAE backbone reaction previously described herein, with an Ac:Am ratio of 1.8:1 and various branching percentages (legend) followed by ether purification and DMSO resuspension as previously described here. This resulted in BPBAE 9,8-91 in a single reaction step. BPBAE 9,8-91 was then complexed with eGFP-N1 plasmid at various w / w (x-axis) and dosed to B16F10 cells in vitro.250 ng DNA / well. 5k cells / well. %Viability assessed via CTG. REFERENCES

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[0404] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. A nanoparticle comprising a branched poly(beta-amino ester) (PBAE) comprising a backbone having a ratio of diacrylate monomers and triacrylate monomers or diacrylate and tetraacrylate monomers, an amine sidechain, and an amine endgroup, and a nucleic acid.

2. The nanoparticle of claim 1, wherein the diacrylate monomer is selected from: ;2); onsisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

3. The nanoparticle of claim 2, wherein the diacrylate monomer is: or4. The nanoparticle of claim 1 or claim 2, wherein the triacrylate monomer or tetraacrylate monomer is selected from: ;O ; an integer selected from the group consisting of 1, 2, 3, 4,5. The nanoparticle of claim 4, wherein the triacrylate monomer or tetraacrylate monomer is selected from:

6. The nanoparticle of any one of claims 1 to 5, wherein the amine sidechain monomer is selected from:OH(S3);OH(S4);7. The nanoparticle of claim 6, wherein the amine sidechain monomer is selected from: O O OH(S91).

8. The nanoparticle of any one of claims 1 to 7, wherein the amine end group is selected from:NH2 (E1); NH2 (E2);; andNH (E39); and H N N NH2(E63). 1 to 9, wherein the branched PBAE isthe branched PBAE comprises 9,8-91-39.

12. The nanoparticle of any one of claims 1 to 11, wherein the nucleic acid is selected from DNA, mRNA, and siRNA.

13. The nanoparticle of any one of claims 1 to 12, comprising a polymer-to-nucleic acid weight-to-weight ratio (w / w) having a range from about 10:1 to about 40:

1.

14. The nanoparticle of any one of claims 1 to 13, wherein the nanoparticle has a percent branching having a range from about 1% to about 60%.

15. The nanoparticle of any one of claims 1 to 14, wherein the nanoparticle has an acrylate:amine (Ac:Am) ratio of 2.2:1 or 2.4:

1.

16. The nanoparticle of any one of claim 1 to 15, wherein the nanoparticle has a size between about 10 nm to about 150 nm in diameter.

17. The nanoparticle of claim 16, wherein the nanoparticle has a size of about 50 nm.

18. A method for treating a disease, condition, or disorder, the method comprising administering a therapeutically effective amount of a nanoparticle of any one of claims 1 to 18 to a subject in need of treatment thereof.

19. The method of claim 18, wherein the disease, condition, or disorder is selected from a cancer, an autoimmune disease, an ocular disease, and a genetic disease or disorder.

20. The method of claim 27, wherein the cancer comprises melanoma or hepatocellular carcinoma.

21. A method for transfecting one or more cells, the method comprising contacting the one or more cells with a nanoparticle of any one of claims 1 to 18.

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